This invention relates to a DNA base sequencer, or an apparatus for determining the base sequences of DNA. More particularly, this invention relates to an apparatus with which the base sequences of DNA can be determined with a plurality of fluorescence markers in an efficient and rapid manner.
Gel electrophoresis is practiced extensively as a technique for determining the base sequences of DNA and other proteins. Conventionally, the sample to be subjected to electrophoresis is labelled with a radioisotope for analysis but this method has had the problem of being painstaking and time-consuming. Furthermore, the use of radioactive substances always calls for utmost safety and management and analysis cannot be performed in areas other than facilities that clear certain regulations. Under the circumstances, a method that uses fluorophores to label the sample and which detects fluorescences as emitted upon irradiation with light is being reviewed.
In this method, fluorophore-labelled DNA fragments are caused to migrate through a gel, and a light excitation portion and a photodetector are provided for each electrophoresis track in an area 5-20 cm below the start point of electrophoresis. The DNA fragments are assayed as they pass through the line connecting the light excitation portion and the photodetector. A typical procedure of the method is described below. First, using as a template the DNA chain to be determined for its base sequence, DNAs of various lengths with known terminal base species are replicated by a method involving an enzymatic reaction (the dideoxy method). Then, the replicated DNAs are labelled with a fluorophore. Stated more specifically, there are prepared a group of adenine (A) fragments, a group of cytosine (C) fragments, a group of guanine (G) fragments and a group of thymine (T) fragments, all being labelled with a fluorophore. A mixture of these fragment groups is injected into separate lane grooves in an electrophoretic gel and, thereafter, a voltage is applied at opposite ends of the gel. Since DNA is a chained polymer with negative charges, it will move across the gel at a rate in inverse proportion to its molecular weight. The shorter the DNA chain (the smaller its molecular weight), the faster will it move and vice versa; this is the principle behind the fractionation of DNA by molecular weight.
With a view to analyzing a greater number of samples on an electrophoresis plate having a limited width, DNA base sequencers have been developed that share a common lane for electrophoresing a plurality of samples (e.g. bases) labelled with different fluorescent dyes so that they can be detected by differentiation with the initial color labels.
An example of such apparatus is shown in FIG. 13. An electrophoresis plate 74 comprises a gel (typically a polyacrylamide gel) held between two glass plates. The electrophoresis plate has an overall thickness of up to about 10 mm but the thickness of the gel electrolyte layer itself is less than about 1 mm. The upper end of the gel electrolyte layer is comb-shaped and located slightly below the upper end of the plate 74. DNA fragments labelled with a plurality of fluorophores (e.g. producing two colors) are injected into grooves 75 corresponding to the teeth of the comb.
In the apparatus shown in FIG. 13, a laser beam emitted from a light source 70 is launched horizontally from one side of the plate 74 at a predetermined point on the gel. As the fluorophore-labelled DNA fragments migrating through the gel pass through the irradiated region, they will fluoresce successively. The horizontal position of fluorescence emission tells the species of a particular terminal base, the time difference from the start of migration tells the length of a particular fragment, and the emission wavelength identifies the sample under assay. A prism 92 is provided adjacent and close to tete-a-tete a principal surface of the electrophoresis plate; a filter 94 composed of an upper and a lower segment is provided adjacent the prism 92; sensor A 96a for receiving fluorescence at one wavelength (.lambda.1) and sensor B 96b for receiving fluorescence at another wavelength (.lambda.2) are provided behind the filter 94; and an imaging lens 98 is provided between the filter 94 and the sensor array. Shown by 100 and 102 in FIG. 13 are an upper buffer tank and a lower buffer tank, respectively.
FIG. 14 shows schematically the principle for differentiating fluorescence of one color from fluorescence of another color. FIG. 15 shows characteristic curves for the fluorescence intensities of two markers, one emitting fluorescence at wavelength .lambda.1 and the other emitting fluorescence at wavelength .lambda.2. FIG. 16 shows corresponding characteristic curves for the fluorescence intensities as obtained after transmission through the filters. Turning back to FIG. 14, if sample 76 labelled with a fluorescent dye emitting at wavelength .lambda.1 crosses a point of emission, fluorescence will be generated that has the intensity shown in FIG. 15. The emitted fluorescence at wavelength .lambda.1 is launched into prism 92, refracted by it and admitted into filter A 94a for receiving fluorescence at wavelength .lambda.1 and filter B 94b for receiving fluorescence at wavelength .lambda.2. The fluorescence at wavelength .lambda.1 that has been admitted into filter B 94b is cut off and incapable of transmission through this filter. Therefore, the fluorescence at wavelength .lambda.1 that has passed through filter A 94a is focused by the imaging lens 98 to form an image on sensor A 94a for subsequent detection. The fluorescence having passed through filter A 94a has substantially the same transmittance as the fluorescence having passed through filter B 94b (see FIG. 16).
The apparatus described above has various drawbacks such as the complexity of the optics, the need to employ more than one sensor, and the lower intensity of fluorescence due to light separation by the prism.
FIG. 17 shows another known example of the DNA base sequencer that employs labelling with two fluorescent dyes emitting at different wavelengths. In the apparatus, a light source 70 positioned in front of an electrophoresis plate 74 emits laser light 90 toward a labelled sample 76 and the resulting fluorescence is received on the same side of the electrophoresis plate. The fluorescence passes through a disk-shaped filter 104 consisting of two segments and is focused by an imaging lens 106 to form an image on a photomultiplier 108. Since the disk-shaped filter 104 is rotating, fluorescence at wavelength .lambda.1 passes through the filter alternately with fluorescence at wavelength .lambda.2. FIG. 18 is a timing chart illustrating how the two fluorescences emitting at wavelengths .lambda.1 and .lambda.2 are detected by means of the rotating filter 104.
A problem with the apparatus shown in FIG. 17 is that it requires a mechanism for rotating the filter and, hence, involves a complicated construction. In addition, all electrophoresis lanes are detected by scanning the entire part of the optics but, then, the detection speed is unavoidably slow because the number of revolutions of the filter has to satisfy a certain relationship with the timing of scanning.